The mechanism by which insulin is able to activate pyruvate dehydrogenase in rat adipose tissue will be investigated. This activation process is of special interest because pyruvate dehydrogenase is present within the confines of the inner mitochondrial membrane, yet insulin apparently need not enter fat cells in order to exert its effect. Therefore, it is postulated that a cytoplasmic messenger mediates this effect of insulin. It is possible that many and perhaps all actions of insulin are mediated by cytoplasmic messengers and the investigation of this system may therefore offer a broad insight into the mechanism of action of this hormone. Pyruvate dehydrogenase activity is regulated by a phosphorylation-dephosphorylation cycle with only the dephospho-form active. Experiments carried out during the initial years of this project suggest that insulin causes a greater fraction of the enzyme to be present in the active form by increasing the activity of the pyruvate dehydrogenase phosphate phosphatase. No evidence was obtained for any action of insulin on the kinase reaction. Further experiments are planned to detail our knowledge of how insulin activates the phosphatase. The assay currently in use for this action of insulin will be sought. Cytoplasmic messengers for insulin will then be sought by using their effect on the phosphatase as the assay system. If active extracts from insulin-treated cells can be obtained, attempts will be made to isolate and identify the active substance.